The long-term goal of this project is to characterize structural, biochemical and pharmacological properties of conopeptides that target G-protein coupled receptors (GPCRs). In the proposed research, we will focus on studying structure-activity-relationships of a unique analgesic conopeptide, Contulakin-G, targeting neurotensin receptors (NTRs). Contulakin-G is a 16-residue peptide isolated from venom of Conus geographus. This peptide shares the C-terminal sequence similarity with an endogenous neuropeptide, neurotensin (NT), but it also contains an unusual posttranslational modification: O-glycosylation of a Thr residue. A unique pharmacological property of Contulakin-G is that this very potent analgesic compound is a low-affinity, non-desensetizing agonist for neurotensin receptors. In stark contrast to Contulakin-G, NT is 600-fold less potent as analgesic, but it is a high-affinity agonist that easily desensitizes NTRs. Results with non-glycosylated Contulakin-G suggested that such profound mechanistic differences between Contulakin-G and neurotensin can be accounted for by the presence of both, glycosylation and distinct N-terminal sequences. We propose to systematically explore structural differences between Contulakin-G and neurotensin that determine a unique pharmacological profile of this conopeptide. Specific aims of this proposal include: (1) chemical synthesis of Contulakin-G and neurotensin analogs varying in the peptidic and glycosyl structures, (2) characterization of in vitro pharmacological properties of Contulakin-G analogs, (3) assessment of analgesic properties of the synthesized ananlogs and their mechanism of analgesia, (4) discovery of novel members of the same gene family that Contulakin-G belongs to. Results from the research will allow to better define structural determinants of unique analgesic properties of Contulakin-G, as well as to test a hypothesis that the potent analgesia produced by this glycopeptide can, at least in part, be accounted for by its ability to not desensitize neurotensin receptor. The long-term outcomes of this proposal may lead to development of novel compounds targeting GPCRs.